Not applicable.
(1) Field of the Invention
The present invention relates to a process for detecting and enumerating Campylobacter jejuni in an environmental sample. The present invention further relates to a process which can distinguish antibiotic resistant strains of Campylobacter jejuni from wild-type strains, in particular, antibiotic-resistant strains resistant to high levels of ciprofloxacin. Both processes relate to PCR primers which flank a target sequence unique to Campylobacter jejuni in combination with one or more dual-labeled oligonucleotide probes complementary to the target sequence wherein the dual-labeled probes enable detection of PCR amplification by fluorescence detection means.
(2) Description of Related Art
Campylobacter spp. is the leading cause of gasterial enteritis in the United States, affecting millions of people annually. Of the people affected, Campylobacter jejuni represents the etiological agent for a large proportion of the clinical cases of Campylobacter infections in human patients. Campylobacter is a gram negative microaerophilic pathogen of both humans and animals. In humans, Campylobacter infection is characterized by acute diarrheal disease, and more recently, has been associated with Guillain Barre Syndrome, a peripheral neuropathy characterized by limb weakness, and other neurological and systemic sequelae (Hughs et al., J. Infect. Dis. 176(Suppl. 2): S92-S98 (1997); Blaser, J. Infect. Dis. 176(Suppl.): S103-S105 (1997)).
Erythromycin, fluoroquinolones, and tetracyclines are classes of antibiotics that are most frequently used to treat Campylobacter infections (Blaser, J. Infect. Dis. 176(suppl. 2): S103-S105 (1997); Altkreuse et al., Emerg. Infect. Dis. 5: 28-35 (1999)). While these antibiotic treatments are effective at controlling bacterial enteritis, increasingly, antibiotic resistant strains of Campylobacter jejuni are being uncovered (Gaudreau et al., Antimicrob. Agents Chemother. 42: 2106-2108 (1998); Gaunt et al., J. Antimicrob. Chemother. 37: 747-757 (1996); Moore et al., Vet. Rec. 138: 306-307 (1996); Lee et al., Internatl. J. Food Microbiol. 24: 161-170 (1994); Taylor et al., Antimicrob. Agents Chemother. 32: 1107-1112 (1988); Tenover et al., Antimicrob. Agents Chemother. 27: 37-41 (1985)). The appearance of antibiotic resistant strains of Campylobacter jejuni is a public health concern. Therefore, much attention has been drawn to Campylobacter jejuni strains which are resistant to fluoroquinolones, a class of antibiotics most frequently used for treating bacterial enteritis of which ciprofloxacin is an example.
The predominant mechanism for high-level ciprofloxacin resistance (MIC equal to or greater than 16 xcexcg/ml) in Campylobacter jejuni appears to be a C to T nucleotide transition in amino acid codon 86 in the quinolone resistance determining region (QRDR) of the gyrA gene. The gyrA gene encodes one subunit of DNA gyrase, which is the target for fluoroquinolone antibiotics. The amino acid codon 86 mutation results in a threonine to isoleucine amino acid substitution in the functional gyrA protein (Wang et al., Antimicrob. Agents Chemother. 37: 457-463 (1993); Charvalos et al., J. Clin. Lab. Anal. 10: 129-133 (1996); Husmann et al., J. Clin. Microbiol. 35: 2398-2400 (1997); Gibreel et al., Antimicrob. Agents Chemother. 42: 3276-3278 (1998); Ruiz et al., Microbiol. Immunol. 42: 223-226 (1998)). Ciprofloxacin susceptibility testing of Campylobacter is commonly performed using standard methods such as broth or agar dilution (Charvalos et al., ibid.; Gaunt et al., ibid.; Gaudreau et al., ibid.; Ruiz et al., ibid.) which are tedious and time consuming methods.
In order to conduct adequate epidemiological studies to determine the sources of Campylobacter jejuni in the agricultural environment, a sensitive diagnostic test is desirable. The appropriate test would allow execution of surveillance programs to monitor contamination of food and the environment in a timely manner. Therefore, the test most appropriate is a test of high sensitivity, specificity, and short turn-around time.
Currently, the standard method for isolation and identification of Campylobacter jejuni involves a pre-enrichment step in supplemental broth and incubation for between about 24 and 48 hours under suitable growth conditions. Next, there follows an isolation step by subculturing the enrichment broth onto Campylobacter selective media and incubation for an additional 24 to 72 hours. The identity of isolated colonies of Campylobacter from selective media can be confirmed using biochemical tests or polymerase chain reaction (PCR) methods. Antibiotic susceptible or resistant Campylobacter strains can be identified using particular susceptibility/resistance testing which requires an additional 24 hours for completion. Thus, to identify antibiotic-resistant strains of Campylobacter jejuni in an environmental sample requires an average of four and a half days to complete.
Therefore, there is a need for a test of improved sensitivity, and specificity, and further of short turn-around time that would enable detection and enumeration of Campylobacter jejuni in environmental samples. With the appearance of strains which are antibiotic resistant, there is a further need for a rapid and sensitive test that will detect these antibiotic-resistant strains in an environmental sample.
The present invention provides a process for detecting and enumerating Campylobacter jejuni in an environmental sample. The present invention further provides a process which can distinguish antibiotic resistant strains of Campylobacter jejuni from wild-type strains, in particular, antibiotic-resistant strains resistant to high levels of ciprofloxacin. Both processes use PCR primers which flank a target sequence unique to Campylobacter jejuni in combination with one or more dual-labeled oligonucleotide probes complementary to the target sequence wherein the dual-labeled probes enable detection of PCR amplification by fluorescence detection means.
Therefore, the present invention provides a process for detecting and enumerating Campylobacter jejuni in a sample, the process comprising: (a) providing in a PCR reaction mixture a sample suspected to contain a target nucleic acid sequence that is unique to Campylobacter jejuni, a first oligonucleotide PCR primer and a second oligonucleotide PCR primer which hybridize to opposite strands of the target nucleic acid sequence and flank the target nucleic acid sequence for PCR amplification of the target nucleic acid sequence, each of four deoxynucleoside triphosphates selected from the group consisting of adenosine, guanosine, thymidine, cytosine, and analogs thereof, a nucleic acid polymerase having a 5xe2x80x2 to 3xe2x80x2 exonuclease activity and lacking 3xe2x80x2 to 5xe2x80x2 exonuclease activity, and an oligonucleotide probe blocked against chain extension at its 3xe2x80x2 end and labeled at its 5xe2x80x2 with an energy transfer donor fluorophore and labeled at its 3xe2x80x2 end with an energy transfer acceptor fluorophore wherein the oligonucleotide probe is complementary to the target nucleic acid; (b) amplifying the target nucleic sequence in the sample under suitable PCR reaction mixture temperature conditions by a repetitive series of PCR thermal cycling steps comprising: (1) denaturing the target nucleic acid sequence into opposite strands; (2) hybridizing the first and second oligonucleotide PCR primers and the oligonucleotide probe to the denatured strands, and (3) extending the hybridized primers with the four deoxynucleoside triphosphates and the nucleic acid polymerase, and producing 5xe2x80x2 fluorophore and 3xe2x80x2 fluorophore labeled nucleotide fragments during the extension phase by the 5xe2x80x2 to 3xe2x80x2 exonuclease activity of the nucleic acid polymerase on the oligonucleotide probe annealed to the denatured strands; (c) following amplification of the target nucleic acid sequence by one or more series of the thermal cycling steps, spectrophotometrically detecting and measuring the amount of fluorescence of the 5xe2x80x2 fluorophore labeled nucleotide fragments wherein the fluorescence indicates the sample contains the Campylobacter jejuni and wherein the amount of fluorescence is proportional to the number of Campylobacter jejuni in the sample.
In a preferred embodiment of the present invention, the target nucleic acid sequence is a nucleotide sequence from QRDR of gyrA, 16S rDNA, or flaA/flaB of Campylobacter jejuni. In particular, wherein the target nucleic acid sequence is the nucleotide sequence in SEQ ID NO:10 and wherein the first and second oligonucleotide PCR primers and the oligonucleotide probe are complementary to the target nucleic acid sequence. In a most preferred embodiment, the first oligonucleotide primer has the nucleotide sequence set forth in SEQ ID NO:3 and the second oligonucleotide PCR primer has the nucleotide sequence set forth in SEQ ID NO:4 and the oligonucleotide probe has the nucleotide sequence set forth in SEQ ID NO:5.
The present invention further provides for a process for detecting and enumerating Campylobacter jejuni wherein the sample comprises a culture from which an environmental sample containing the Campylobacter jejuni is cultivated; or wherein the sample comprises the Campylobacter jejuni which is isolated from an environmental sample by immunomagnetic separation; or wherein the sample comprises amplified DNA produced by a PCR using a first primer selected from SEQ ID NO:3 and SEQ ID NO:8 and a second primer selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:9.
The present invention further provides a process for detecting in a sample antibiotic-resistant Campylobacter jejuni and wild-type Campylobacter jejuni, in particular wherein the antibiotic-resistant Campylobacter jejuni is resistant to ciprofloxacin, the process comprising: (a) providing in a PCR reaction mixture a sample suspected to contain a target nucleic acid sequence that is unique to Campylobacter jejuni, a first oligonucleotide PCR primer and a second oligonucleotide PCR primer which hybridize to opposite strands of the target nucleic acid sequence and flank the target nucleic acid sequence for PCR amplification of the target nucleic acid sequence, each of four deoxynucleoside triphosphates selected from the group consisting of adenosine, guanosine, thymidine, cytosine, and analogs thereof, a nucleic acid polymerase having a 5xe2x80x2 to 3xe2x80x2 exonuclease activity and lacking 3xe2x80x2 to 5xe2x80x2 exonuclease activity, a first oligonucleotide probe blocked against chain extension at its 3xe2x80x2 end and labeled at its 5xe2x80x2 with a first energy transfer donor fluorophore and labeled at its 3xe2x80x2 end with an energy transfer acceptor fluorophore, and a second oligonucleotide probe blocked against chain extension at its 3xe2x80x2 end and labeled at its 5xe2x80x2 with a second energy transfer donor fluorophore and labeled at its 31 end with an energy transfer acceptor fluorophore, wherein the first donor fluorophore emits fluorescent light of a different wavelength than the second donor fluorophore, and wherein the first oligonucleotide probe is complementary to the target nucleic acid from the wild-type Campylobacter jejuni and the second oligonucleotide probe is complementary to the target from the antibiotic-resistant Campylobacter jejuni; (b) amplifying the target nucleic sequence in the sample under suitable PCR reaction mixture temperature conditions by a repetitive series of PCR thermal cycling steps comprising: (1) denaturing the target nucleic acid sequence into opposite strands; (2) hybridizing the first and second oligonucleotide PCR primers and the first and second oligonucleotide probes to the denatured strands, and (3) extending the hybridized primers with the four deoxynucleoside triphosphates and the nucleic acid polymerase, and producing 5xe2x80x2 fluorophore and 3xe2x80x2 fluorophore labeled nucleotide fragments during the extension phase by the 5xe2x80x2 to 3xe2x80x2 exonuclease activity of the nucleic acid polymerase on the oligonucleotide probe annealed to the denatured strands; (c) following amplification of the target nucleic acid sequence by one or more series of the thermal cycling steps, spectrophotometrically detecting and measuring an amount of fluorescence of the 5xe2x80x2 fluorophore labeled nucleotide fragments wherein fluorescence of the first fluorophore indicates the sample contains wild-type Campylobacter jejuni and fluorescence of the second fluorophore indicates the sample contains antibiotic-resistant Campylobacter jejuni and wherein a ratio of the first and second fluorescence is proportional to the ratio of the wild-type and the antibiotic-resistant Campylobacter jejuni in the sample.
In a preferred embodiment of the process of the present invention relating to allelic discrimination, the target nucleic acid sequence is a nucleotide sequence from QRDR of gyrA, 16S rDNA, or flaA/flaB of Campylobacter jejuni. In particular, wherein the target nucleic acid sequence is the nucleotide sequence in SEQ ID NO:10 and wherein the first and second oligonucleotide PCR primers and the oligonucleotide probe are complementary to the target nucleic acid sequence. In a most preferred embodiment, the first oligonucleotide primer has the nucleotide sequence set forth in SEQ ID NO:3 and the second oligonucleotide PCR primer has the nucleotide sequence set forth in SEQ ID NO:4 and wherein the first oligonucleotide probe has the nucleotide sequence set forth in SEQ ID NO:6 and the second oligonucleotide probe has the nucleotide sequence set forth in SEQ ID NO:7.
The present invention further provides for a process for distinguishing antibiotic-resistant Campylobacter jejuni from antibiotic-susceptible Campylobacter jejuni wherein the sample comprises a culture from which an environmental sample containing the Campylobacter jejuni is cultivated; or wherein the sample comprises the Campylobacter jejuni which is isolated from an environmental sample by immunomagnetic separation; or wherein the sample comprises amplified DNA produced by a PCR using a first primer selected from SEQ ID NO:3 and SEQ ID NO:8 and a second primer selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:9.
The present invention further provides a kit for detecting Campylobacter jejuni in a sample comprising in one or more containers: (a) a first oligonucleotide primer; and (b) a second oligonucleotide primer, wherein the first and second oligonucleotides primers hybridize to opposite strands of a target sequence unique to Campylobacter jejuni and flank the target nucleic acid sequence for PCR amplification of the target nucleic acid sequence. In a preferred embodiment of the present invention, the kit further comprises an oligonucleotide probe blocked against chain extension at its 3xe2x80x2 end and labeled at its 5xe2x80x2 with an energy transfer donor fluorophore and labeled at its 3xe2x80x2 end with an energy transfer acceptor fluorophore wherein the oligonucleotide probe is complementary to the target nucleic acid sequence and wherein the oligonucleotide probe enables fluorescence detection and enumeration of the Campylobacter jejuni. In a most preferred embodiment, the target nucleic acid sequence is a nucleotide sequence from QRDR of gyrA, 16S rDNA, or flaA/flaB of Campylobacter jejuni. In particular, a kit wherein the first primer has the nucleotide sequence set forth in SEQ ID NO:3, the second oligonucleotide PCR primer has the nucleotide sequence set forth in SEQ ID NO:4, and the oligonucleotide probe has the nucleotide sequence set forth in SEQ ID NO:5.
In an embodiment further still, the kit comprises in one or more containers at least one of an optimized buffer for the reaction, a control nucleic acid comprising the target nucleic acid sequence, nucleoside triphosphates selected from the group consisting of adenosine, guanosine, thymidine, cytosine, and analogs thereof, a polymerase, a primer with the sequence set forth in SEQ ID NO:8, or a primer with the sequence set forth in SEQ ID NO:9.
The present invention further provides a kit for detecting antibiotic-resistant and wild-type Campylobacter jejuni in a sample, in particular wherein the antibiotic-resistant Campylobacter jejuni is resistant to ciprofloxacin, comprising in one or more containers: (a) a first oligonucleotide primer; (b) a second oligonucleotide primer; (c) a first oligonucleotide probe; and (d) a second oligonucleotide probe, wherein the first and second oligonucleotide primers hybridize to opposite strands of a target sequence unique to Campylobacter jejuni and flank the target nucleic acid sequence for PCR amplification of the target nucleic acid sequence, wherein the first and second oligonucleotide probes are blocked against chain extension at its 3xe2x80x2 end and labeled at the 5xe2x80x2 with a an energy transfer donor fluorophore and labeled at the 3xe2x80x2 end with an energy transfer acceptor fluorophore, wherein the donor fluorophore on the first oligonucleotide probe emits fluorescent light of a different wavelength than the donor fluorophore on the second oligonucleotide probe, and wherein the first oligonucleotide probe is complementary to the target nucleic acid from the wild-type Campylobacter jejuni and the second oligonucleotide probe is complementary to the target from the antibiotic-resistant Campylobacter jejuni. 
In a preferred embodiment, the target nucleic acid sequence is a nucleotide sequence from QRDR of gyrA, 16S rDNA, or flaA/flaB of Campylobacter jejuni. Preferably, wherein the first primer has the nucleotide sequence set forth in SEQ ID NO:3, the second oligonucleotide PCR primer has the nucleotide sequence set forth in SEQ ID NO:4, the first oligonucleotide probe has the nucleotide sequence set forth in SEQ ID NO:6, and the second oligonucleotide probe has the nucleotide sequence set forth in SEQ ID NO:7.
In an embodiment further still, the kit comprises in one or more containers at least one of an optimized buffer for the reaction, a control nucleic acid comprising the target nucleic acid sequence, nucleotide triphosphates selected from the group consisting of adenosine, guanosine, thymidine, cytosine, and analogs thereof, a polymerase, a primer with the sequence set forth in SEQ ID NO:8, or a primer with the sequence set forth in SEQ ID NO:9.
The present invention further provides a labeled nucleic acid probe for detecting Campylobacter jejuni DNA comprising the nucleotide sequence 5xe2x80x2-TTTGCTTCAGTATAACGCATCGCAGC-3xe2x80x2 (SEQ ID NO:5) and a labeled nucleic acid probe for detecting Campylobacter jejuni DNA resistant to an antibiotic comprising the nucleotide sequence 5xe2x80x2-CCACATGGAGATATAGCAGTTTATGATGC-3xe2x80x2 (SEQ ID NO:7). In a preferred embodiment, the probe is labeled at its 5xe2x80x2 end with an energy transfer donor fluorophore and labeled at its 3xe2x80x2 end with an energy transfer acceptor fluorophore. Preferably, the probe is blocked against chain extension at its 3xe2x80x2 end.
In any one of the above embodiments of the present invention, the fluorophore is preferably selected from the group consisting of fluorescein, 5-carboxyfluorescein (FAM), 2xe2x80x2,7xe2x80x2-dimethoxy-4xe2x80x2,5xe2x80x2-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,Nxe2x80x2,Nxe2x80x2-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4xe2x80x2-dimethylaminophenylazo) benzoic acid (DABCYL), tetrachloro-6-carboxy-fluorescein (TET), VIC, and 5-(2xe2x80x2-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).
Finally, the present invention provides a process for detecting Campylobacter jejuni in a sample, the process comprising: (a) providing in a PCR reaction mixture a sample suspected to contain a target nucleic acid sequence that is unique to Campylobacter jejuni, a first oligonucleotide PCR primer and a second oligonucleotide PCR primer which hybridize to opposite strands of the target nucleic acid sequence and flank the target nucleic acid sequence for PCR amplification of the target nucleic acid sequence, each of four deoxynucleoside triphosphates selected from the group consisting of adenosine, guanosine, thymidine, cytosine, and analogs thereof, and a nucleic acid polymerase having a 5xe2x80x2 to 3xe2x80x2 exonuclease activity and lacking 3xe2x80x2 to 5xe2x80x2 exonuclease activity; (b) amplifying the target nucleic sequence in the sample under suitable PCR reaction mixture temperature conditions to provide a detectable amount of amplified target nucleic acid sequence by a repetitive series of PCR thermal cycling steps comprising: (1) denaturing the target nucleic acid sequence into opposite strands; (2) hybridizing the first and second oligonucleotide PCR primers and the oligonucleotide probe to the denatured strands, and (3) extending the hybridized primers with the four deoxynucleoside triphosphates and the nucleic acid polymerase; and (c) detecting the amplified target nucleic acid sequence.
Preferably, the target nucleic acid sequence is a nucleotide sequence from QRDR of gyrA, 16S rDNA, or flaA/flaB of Campylobacter jejuni. In particular, wherein the target nucleic acid sequence is the nucleotide sequence in SEQ ID NO:10 and wherein the first and second oligonucleotide PCR primers are complementary to the target nucleic acid sequence. Most particularly, the first oligonucleotide primer has the nucleotide sequence set forth in SEQ ID NO:3 and the second oligonucleotide PCR primer has the nucleotide sequence set forth in SEQ ID NO:4.
In a further embodiment of the above process, a labeled probe selected from the group consisting of SEQ ID NO:6 and SEQ ID NO:7 is hybridized to the amplified target nucleic acid sequence to determine whether the Campylobacter jejuni is antibiotic-resistant.
Therefore, it is an object of the present invention to provide a process for detecting and enumerating Campylobacter jejuni in an environmental sample.
It is also an object of the present invention to provide a process for detecting in an environmental sample antibiotic-resistant Campylobacter jejuni from antibiotic susceptible Campylobacter jejuni. 
It is further an object of the present invention to be able to detect and quantify Campylobacter jejuni in a sample without the need to purify genomic DNA, and further to directly detect Campylobacter jejuni in an environmental sample, which includes but is not limited to, fecal matter, food, soil, and water, without the need to culture the microorganism.